BackgroundOur previous study determined that tetrandrine (Tet) has an inhibitory effect on the proliferation of human Tenon capsule fibroblasts ( TCFs ) in vitro,but its mechanism is poorly understood.ObjectiveThis study was to investigate the effect mechanism of Tet on human TCFs.MethodsHuman TCFs were isolated and cultured from scleral tissue of donor using explant technique.The cells were identified by vimentin antibody staining and morphology.The third generation of cells were seeded in the culture plate at the density of 1 × 105 cells/ml.Twenty-four hours after inoculation,the Tet of 1 × 10-5 mol/L was added in the well of culture plate,and the cells cultured only in 1640 medium served as the control group.The apoptosis of the cells was assessed by TUNEL,and the expressions of bax,bel-2,transforming growth factor-β2 (TGF-β2 ) in TCFs were detected using immunochemistry.Results The cultured cells showed the features of the fibroblasts in shape with the positive response for vimentin.A number of TUNEL positive cells were seen in Tet group and no TUNEL positive response was found in control group.The expression levels (A value) of bax,bcl-2 and TGF-~ protein in TCFs were 0.577 ± 0.009,0.430±0.012 and 0.341 ±0.017 in Tet group,and those in control group were 0.320±0.015,0.819±0.021 and 0.624±0.014 respectively,showing statistically significant differences between two groups( t =33.277,-35.356,-28.093,P＜0.01 ).Conclusions Tet suppresses the proliferation of human TCFs through up-regulating the expression of bax and down-regulating the expressions of bcl-2 and TGF-β2 in vitro.

BACKGROUNDPrevious studies have showed that photooxidative stress can lead to down-modulation of nuclear factor-kappa B (NF-kappaB) activity causing apoptosis of cultured photoreceptor cells. This study aimed at investigating whether NF-kappaB was involved in photoreceptor cells apoptosis induced by N-methyl-N-nitrosourea (MNU) in rats.

METHODSA single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats. At different intervals after MNU treatment, the animals were sacrificed. Retinal damage was examined by a light microscope. The apoptotic index of the photoreceptor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). NF-kappaB was analysed by Western blot and Transcriptin Factor Assay Kits.

RESULTSThe pyknosis of the photoreceptor nuclei and the disorientation of the outer segment of the photoreceptor layer was seen after MNU treatment for 24 hours. The outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. Photoreceptor cells apoptosis reached the peaked value at 24 hours. In apoptotic cascade, the protein levels of NF-kappaB p65 were only detected after MNU treatment for 12 and 24 hours in the nucleus. Conversely, the amounts of IkappaBalpha were markedly increased in the cytoplasm as well as in the nucleus. The activity of NF-kappaB p65 in the nucleus was down-modulated in the end.

CONCLUSIONSMNU-induced photoreceptor cell destruction was attributed to the apoptotic process by down-regulating the activation of NF-kappaB p65.

Animals ; Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Female ; I-kappa B Proteins ; analysis ; physiology ; Methylnitrosourea ; toxicity ; NF-KappaB Inhibitor alpha ; NF-kappa B ; analysis ; physiology ; Photoreceptor Cells ; chemistry ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; pathology

OBJECTIVETo observe the changes of nuclear factor-kappa B (NF-kappaB) in the course of N-methyl-N-nitrosourea (MNU)-induced apoptosis of rat retinal photoreceptor cells and investigate the mechanism of MNU-induced retinal damage.

METHODSA single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats, which were sacrificed at different intervals after MNU treatment. The retinal damage was examined with optical microscopy and photoreceptor cell apoptosis detected by TUNEL assay. Western blotting was performed to analyze the changes in NF-kappaB.

RESULTSPyknosis of the photoreceptor cell nuclei and disorientation of the outer segment of the photoreceptor layer was observed 24 h after MNU treatment, and the outer nuclear layer and photoreceptor layer were almost completely lost on day 7. Photoreceptor cell apoptosis peaked at 24 h, and in the apoptotic cascade, NF-kappaB p65 protein was only detected 12 and 24 h after MNU treatment, whereas the amount of I kappa B alpha, in contrast, markedly increased in the cytoplasm as well as in the nuclei.

CONCLUSIONMNU-induced retinal damage might be mediated through the signaling pathway of NF-kappaB/I kappa B alpha.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Female ; I-kappa B Proteins ; metabolism ; In Situ Nick-End Labeling ; Methylnitrosourea ; toxicity ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retinal Diseases ; chemically induced ; metabolism ; pathology

OBJECTIVETo investigate the change of expression level of metastasis suppressor gene Kiss-1 in the colorectal cancer cell line SW480 after radiation, and to determine its association with the proliferation and apoptosis of SW480 cells.

METHODSSW480 cells were divided into control group (0 Gy) and study groups (2, 4, 6, 8 Gy). Cells in the study groups were irradiated by 6-MV X-ray radiation for 48 hours. Immunohistochemistry and real-time PCR methods were used to investigate the influence of radiation on Kiss-1 gene expression of SW480. Colony formation assay was used to detect the proliferation of SW480. Flow cytometry-Annexin- V/PI assay was used to observe the change of the apoptosis rate.

RESULTSCompared with the control group, Kiss-1 protein expression increased after radiation of 6, 8 Gy (P<0.05), but no significant changes were observed after radiation of 2, 4 Gy(P>0.05). Kiss-1 gene mRNA level increased after radiation of 2, 4, 6 Gy, while no obvious change was observed for 8 Gy radiation. The apoptosis rates increased for 4, 6, 8 Gy radiation(P<0.05), however, there was no significant difference for 2 Gy radiation (P<0.05).

CONCLUSIONRadiation may increase Kiss-1 gene expression in SW480 cells, which results in decreases proliferation and increases apoptosis in residual surviving cells.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Humans ; Kisspeptins ; genetics ; metabolism ; radiation effects ; RNA, Messenger ; genetics ; X-Rays

OBJECTIVETo study the prevention & treatment effect of cordyceps sinensi on bleomycin-induced pulmonary fibrosis (PF) in mice.

METHODRats were divided into 4 groups. In BM group (model), pulmonary interstitial fibrosis model was established through nasal dripping of 5 mg x kg(-1) bleomycin. In PCT group (prevention & treatment with cordyceps sinensi), cordyceps sinensi was orally administered 48 h before bleomycin dripping. In CT group (treatment with cordyceps sinensi), cordyceps sinensi was orally administered at 14 d after bleomycin dripping, and NC group (normal mice, control). At 28 d, bronchoalveolar lavage fluid (BALF) and lung tissue were collected, right lung received HE staining and masson staining, the change in cell micro-structure was observed under electron microscope, the hydroxyproline (HYP) content in lung tissue of left lung was detected through acidolysis method, and TGF-beta1, IFN-gamma and IL-4 concentration in BALF was detected through ELISA.

RESULTCompared with those in BM group, mouse lung coefficient decreased significantly (P < 0.05), alveolitis and fibrosis degree improved significantly in PCT and CT groups (P < 0.05), HYP content in lung decreased (P < 0.05). TGF-beta1 and 1L-4 content in BALF decreased significantly in each drug intervention group, IFN-gamma content increased significantly (P < 0.05).

CONCLUSIONThe cordyceps sinensi can alleviate bleomycin-induced alveolitis and fibrosis degree in a certain degree. The mechanism of the alleviation is possibly due to its inhibition of TGF-beta1 expression and its regulation of imbalance of the type I and II cytokine in lung tissue.

Animals ; Bleomycin ; Bronchoalveolar Lavage Fluid ; chemistry ; Cordyceps ; chemistry ; Hydroxyproline ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lung ; metabolism ; pathology ; ultrastructure ; Male ; Mice ; Mice, Inbred ICR ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; Random Allocation ; Transforming Growth Factor beta1 ; metabolism

Objective To study influence of the mothers' accompany on pediatric anesthesia during venous revive recovery.Methods 58 cases with intravenous anesthesia pediatric surgery were enrolled and randomly divided into observation group and control group (29 cases in each group), the observation group was accompanied with mother in the anesthesia awaking room, and children in control group were not accompanied with their mothers.Children's restless, psychological status and revive period were observed in the two groups.Results Restless during revive period in observation group was significantly lower than control group (20.7% vs 69.0% ,x2 = 13.66,P ＜0.01) ;Fear,anxiety rate was lower than the control group(20.7% vs 89.7% ,x2 =27.88,P ＜0.01); BP、HR between two groups were significant difference (P ＜ 0.01).Conclusions Mother can significantly reduce anxiety, fear and restless incident rate of pediatric anesthesia.

OBJECTIVETo investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC).

METHODSThe human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-specific phosphatase inhibitor (salubrinal) to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and western blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test.

RESULTSCisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment significantly inhibited the cisplatin (15 mg/ml)-induced apoptosis in SMMC-7721 (control: 50.3 +/- 2.5% vs. 16.3 +/- 2.1%, t = 18.031, P less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05).

CONCLUSIONPhosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Eukaryotic Initiation Factor-2 ; metabolism ; Humans ; Liver Neoplasms ; Phosphorylation

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.
ATP-Binding Cassette, Sub-Family B, Member 1 ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Antibody Specificity ; genetics ; physiology ; Blotting, Western ; CD3 Complex ; immunology ; Chromatography, Gel ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Flow Cytometry ; Humans ; Jurkat Cells ; drug effects ; metabolism ; K562 Cells ; drug effects ; metabolism ; Mice ; Polymerase Chain Reaction ; Radioimmunoassay ; Trifluoperazine ; pharmacology ; Verapamil ; pharmacology

OBJECTIVETo prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration.

METHODSMcAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb.

RESULTSMcAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively.

CONCLUSIONThe McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.

Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; CD3 Complex ; immunology ; Cell Line ; Chromatography, Affinity ; Humans ; Hybridomas ; metabolism ; Jurkat Cells ; K562 Cells

OBJECTIVETo investigate the role of miR-221/222 in inhibiting endoplasmic reticulum stress-induced human hepatocarcinoma cells apoptosis.

METHODmiR-221/222 mimics and inhibitors were used to mimic or block the function of endogenous miR-221/222 respectively. Western blot and flow cytometry were used to test the effects of miR-221/222 on cell cycle and apoptosis under endoplasmic reticulum stress in human hepatocellular carcinoma cells.

RESULTSEndoplasmic reticulum stress resulted in miR-221/222 down-regulation in human hepatocellular carcinoma cells. miR-221/222 mimics and inhibitors inhibited and promoted respectively endoplasmic reticulum stress-mediated p27Kip1 induction. Moreover, p27Kip1 suppression not only resulted in reduction in the fraction of G1 phase cells, but also promoted the endoplasmic reticulum stress-mediated apoptosis in human hepatocellular carcinoma cells.

CONCLUSIONmiR-221/222 were downregulated by endoplasmic reticulum stress in human hepatocellular carcinoma cells, which subsequently protected human hepatocellular carcinoma cells against endoplasmic reticulum stress-induced apoptosis through p27Kip1 regulation.

Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Endoplasmic Reticulum ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; MicroRNAs ; metabolism